A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles

作者：Tang Wenxing; Knox Robert W; Nevzorov Alexander A^*

来源：Journal of Biomolecular NMR, 2012, 54(3): 307-316.

DOI：10.1007/s10858-012-9673-y

• Oriented-sample NMR (OS-NMR)
• Cross-polarization
• Membrane proteins
• Pf1
• Spectroscopic assignment
• Magnetically aligned bicelles
• Mismatched Hartmann Hahn conditions

Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the %26quot;shotgun%26quot; approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the N-15 chemical shift and N-15-H-1 dipolar couplings, as well as spin-correlations between the (i, i +/- A 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an N-15-N-15 correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled %26quot;seed%26quot; spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B (1) fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.

• 出版日期2012-11